In an expanding art field that embraces the discovery of new drugs and combinatorial chemistry used in the preparation of new candidate compounds, it would be especially advantageous to be able to screen a great number of substances by way of a procedure affording high throughput, to the end of observing their physiological impact on animals and on humans. Before testing the efficacy of a drug candidate “partially successful” on animals, the substance should be tested for its potential toxicity in respect of living cells. Conversely, it is the conventional practice that promising compounds are tested almost immediately in extensive studies on animal models, studies which are at the same time lengthy and costly. Moreover, the practice of extensive testing on animals is becoming less and less acceptable culturally in the United States and in Europe. If prospective pharmaceutical compounds are tested to observe their interactions with living cells before studies are conducted on animal models, this can reduce the number of animals needed for subsequent trials by eliminating many of the candidates before the stage of testing on animals is reached.
Current procedures for the analysis of cell-drug interaction afford neither high throughput nor high biological value, due to the limited number of cells and compounds that can be analyzed in a given period of time, the scant practicality of the methods necessary for administering the compounds, and the considerable volumes of the compound required.
Accordingly, efforts have been made to overcome these drawbacks by studying alternative methods for the analysis of interactions between cell and drug, or more generally between biological samples and biologically active agents, such as those indicated below by way of example.
Cell Matrices
Several methods have been described for producing uniformly micro-modelled cell matrices, for example photolithography (Mrksich & Whitesides, Ann. Rev. Biophys. Biomol. Struc. 25:55-78, 1996). According to this method, which uses a glass plate, a photosensitive material and a mask are employed to obtain a plate presenting a matrix of reactive or hydrophilic spots on a surface which by contrast is hydrophobic. The matrix of hydrophilic groups provides a substrate on which to obtain a non-specific and non-covalent bond of certain types of cells, including those of neuronal origin (Kleinfeld et al., J. Neurosci. 8:4098-4120, 1988).
In another method based on specific but non-covalent interactions, photoprinting is used to produce a gold surface presenting spots of laminin, a cell-binding protein normally found in the extracellular matrix (Singhvi et al., Science 264: 696-698, 1994)
A more specific uniform bond can be obtained by crosslinking specific molecules, such as proteins, at reactive sites of the modelled substrate (Aplin & Hughes, Analyt. Biochem. 113: 144-148, 1981).
Another development of an optical system for modelling a substrate and creating reactive spots is based on the use of deep UV rays directed through an optical mask, to obtain active sites consisting in polar silanol groups. These groups make up the spots of the matrix and are modified further by being paired with other reactive groups, as disclosed in U.S. Pat. No. 5,324,591. This optical method of forming uniform cell matrices on a substrate requires fewer steps than the photolithography method, but requires ultraviolet light of high intensity, and suitable light sources are very costly.
In all these methods moreover, the resulting cell matrix is uniform, since the biochemically specific molecules are bound to the chemically micro-modelled matrices. With the photolithography method, the cells bind to matrices of hydrophilic spots and/or to specific molecules attached to the spots which bind the cells. Accordingly, the cells bind to all the spots of the matrix in the same way. With the optical method, the cells bind to matrices containing spots of free amino acid groups by adhesion. There is little or no difference between these spots. Here again, the cells bind to all the spots in the same way and it is possible only to study one given type of cell interaction using these matrices, since any one spot is essentially the same as another.
This type of matrix therefore lacks flexibility as an instrument for the analysis of a single specific variety of cell or interaction. Consequently, the need arises to produce cell matrices that are not uniformly micro-modelled, so as to increase the number of cells or interactions that can be analyzed simultaneously.
International patent applications WO 00/39587 and WO 00/47996 illustrate a system of sensors and methods for preparing matrices, composite or otherwise, of beads or cells ordered randomly on the tips of bundles of optical fibres. Whilst on the one hand the methods described in these applications offer great analytical potential, especially in the case of proteins and nucleic acids, there is the drawback that they allow the experimenter neither to separate nor, much less, to recover populations of interest that may be identified.
Cell Physiology and Fluorescence
Conducting a high throughput assay on many thousands of compounds requires the manipulation in parallel and the treatment of many compounds and of the reagents included in the assay; in addition, there must be a method of identifying and measuring the results of the experiment in the simplest way possible. The more common assays use homogeneous blends of compounds and biological reagents together with at least one marker compound, loaded into a standard 96 or 384-well microtiter plate (Kahl et al., J. Biomol. Scr. 2:3340, 1997). The signals measured from each well, whether emissions of fluorescence, optical density or radioactivity, are integrated with the signal from all the material occupying the well to give a general average of the population of all molecules in the well. This type of assay is commonly termed a homogeneous assay.
As fluorescence is among the systems most widely used, various methods have been developed for generating images of fluorescent cells with a microscope and extracting information on the spatial distribution and the changes occurring over time in these cells. Many of these methods and their applications are described in an article by Taylor et al., Am. Scientist 80: 322-335, 1992.
The proposed methods have been designed and optimized with the preparation of a small number of samples in view so that the distribution, quantity and biochemical profile of fluorescent reporter molecules present in the cells can be measured obtaining a high level of spatial and temporal resolution.
Useful methods of detection include treating the cells with colorants and fluorescent reagents to obtain images and/or genetically modify the cells in such a way that they will produce fluorescent proteins, like modified Green Fluorescent Protein (GFP). The use of GFP in the study of gene expression and the localization of proteins is discussed at length by Chalfic et al., in Science 263: 803-805.
Nonetheless, these methods are complex, costly and slow, and they can be used only to study cells in groups, not individually.
Dielectrophoresis
Dielectrophoresis relates to the physical phenomenon whereby dielectric particles subject to spatially non-uniform d.c. and/or a.c. electric fields undergo a net force directed toward those regions of space characterized by increasing (pDEP) or decreasing (nDEP) field strength. If the strength of the resulting forces is comparable to the force of gravity, it is possible in essence to create an equilibrium of forces enabling the levitation of small particles. The strength, direction and orientation of the dielectrophoretic force are heavily dependent on the dielectric and conductive properties of the body and of the medium in which it is immersed, and these properties in turn vary with frequency.
Studies analyzing the effects of dielectrophoretic forces on microorganisms or biological matter generally (cells, bacteria, viruses, DNA, etc.), and on inorganic matter, have suggested for some time the notion of exploiting these forces as a means of selecting a particular body from a sample containing a plurality of microorganisms, characterizing the physical properties of microorganisms and in general allowing their manipulation.
By way of example, international patent application WO 00/47322 teaches the manipulation of generic “packages” of substances (liquid, solid or gaseous) utilizing dielectrophoretic forces generated between contiguous electrodes of an addressable array. All the same, the method described in this reference is not suitable for conducting a study of high biological value on cells and more generally on microorganisms or parts thereof (DNA and RNA sequences, plasmids, etc.), since on the one hand the “package” is subject to significant voltages to allow its manipulation by dielectrophoresis, and on the other, subject more generally to friction against the reaction surface bearing the array of electrodes.
The prior art embraces another system based on the creation of three-dimensional cell manipulation cages by constructing micro octupoles (T. Schnelle et al., in Biochimica et Biophysica Acta, 1157:127-140); in this instance the cell material is levitated and therefore unaffected by frictional or other mechanical stresses, particularly in the case where the dimensions of the manipulation systems adopted are comparable with those of the particles being manipulated, thus reducing the order of magnitude of the voltages used to create the necessary field distributions when undesirable effects appear (Washizu & Kurosawa, Trans. Ind. Appl. 26:1165-1172, 1990; Washizu et al., Trans. Ind. Appl. 30:835-843, 1994).
However, the structures proposed in literature encounter problems of embodiment when the dimensions of the projected cage approach those of the actual cells (to the end of trapping a single cell). In this instance, the problem consists of aligning the two structures which, assembled one with another on a micrometric scale, make up the octupole.
This particular problem is solved according to international patent application WO 00/69565, which discloses an apparatus and a method for the manipulation of particles (the term “particles” is used hereinafter to denote dielectrophoretically manipulated elements utilized in experiments, be they biological entities, substances compounded with a delivery agent, or both; which of the three cases is intended will be discernible from the context) utilizing closed dielectrophoretic potential cages.
At all events, these latter methods of manipulation based on dielectrophoretic levitation of the material for analysis are limited currently to the separation and/or count of the manipulated particles enabled by recognition of the selfsame particles using suitable sensors of specific type prepared and integrated into the array of electrodes and/or disposed internally or externally of the chamber in which the levitational manipulation takes place. This means that such methods can be used only for particles with intrinsic distinctive characteristics (discriminated on the basis of size, for example) detectable with specific sensors, which must be prepared on a case by case basis.